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Hwy 155, 20 mi. W. of Lake IsabellaS. slopeGrass and Oa

cryptolepis

Carex cryptolepis Mackenzienortheastern sedge;hidden-scale sedge;yellow sedge;small yellow sedgecarex à écailles cachéesCarex cryptolepisalong old logging road in branch valley of French Annie Creek, 3 mi. SW of Copper Harborwet soi

Label-Free Real-Time Microarray Imaging of Cancer Protein–Protein Interactions and Their Inhibition by Small Molecules

A rapid optical microarray imaging approach for anticancer drug screening at specific cancer protein–protein interface targets with binding kinetics and validation by a mass sensor is reported for the first time. Surface plasmon resonance imager (SPRi) demonstrated a 3.5-fold greater specificity for interactions between murine double minute 2 protein (MDM2) and wild-type p53 over a nonspecific p53 mutant in a real-time microfluidic analysis. Significant percentage reflectivity changes (Δ%<i>R</i>) in the SPRi signals and molecular-level mass changes were detected for both the MDM2–p53 interaction and its inhibition by a small-molecule Nutlin-3 drug analogue known for its anticancer property. We additionally demonstrate that synthetic, inexpensive binding domains of interacting cancer proteins are sufficient to screen anticancer drugs by an array-based SPRi technique with excellent specificity and sensitivity. This imaging array, combined with a mass sensor, can be used to study quantitatively any protein–protein interaction and screen for small molecules with binding and potency evaluations

The SNARE Complexes Remain in <i>syx^3–69</i> Mutant Flies at the Restrictive Temperature

<div><p>(A and B) The SDS-resistant complex is not obviously affected in homozygous <i>syx^3–69</i> mutant flies at restrictive temperatures. A representative Western blot shows the syntaxin 1A monomer, the 7S SNARE complex, and the multimeric complex obtained from heads of the wild type (+/+) and the <i>syx^3–69</i> mutant (A). A control for total protein loaded is illustrated by the intensity of tubulin (bottom). Histograms of ratios of the 7S and multimeric complexes to monomer in wild-type (+/+) and <i>syx^3–69</i> mutant flies are shown in (B). Unless specifically noted, the complex-to-monomer ratio is normalized to that of the wild type at room temperature in this and other SNARE complex histograms. The SNARE complex was extracted from flies either at room temperature (∼22 °C) or after exposure to 38 °C for 20 min, as described above [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0050072#pbio-0050072-b021" target="_blank">21</a>].</p> <p>(C) An example of Western blots showing the SNARE complex in the wild-type (+/+), <i>comatose (comt^tp7),</i> and <i>syx^3–69</i> mutant flies. The SNARE complex accumulates in the <i>comt^tp7</i> mutant, likely as a result of a block of the NSF ATPase activity at the restrictive temperature. Note that even though relatively less protein was loaded in the <i>syx^3–69</i> lanes (as judged by the intensity of the syntaxin band), the 7S complex remains in paralyzed <i>syx^3–69</i> flies.</p></div

Conservation and Divergence of Threonine 254 among Different Syntaxin Orthologs

<div><p>(A) Proposed model of SNARE complex assembly and disassembly in a synaptic vesicle cycle (adapted from [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0050072#pbio-0050072-b005" target="_blank">5</a>]). (1) Synaptobrevin forms a partial <i>trans</i> SNARE complex with syntaxin 1A and SNAP-25. (2) By zippering in an N- to C-termini direction, the SNARE proteins form a <i>trans</i> complex and bring the synaptic vesicle close to the plasma membrane. SNARE-mediated synaptic vesicle exocytosis occurs either spontaneously (3) or evoked by Ca²⁺ (4). (5) <i>cis</i> SNARE complexes are thought to be disassembled by NSF ATPase prior to vesicle recycling. ER, endoplasmic reticulum; PM, plasma membrane; SV, synaptic vesicle.</p> <p>(B) Alignment of amino acids (aa) around position T254 in the <i>Drosophila</i> syntaxin 1A or equivalent residues in syntaxin orthologs from a variety of animals, yeast, and the plant <i>Arabidopsis</i>. The top panel shows a cartoon of syntaxin 1A and the region of the alignment. Syntaxins are organized as “plasma membrane” or “intracellular compartments” according to their cellular distributions. With the exception of syntaxin 4, most plasma membrane syntaxins are known to function in presynaptic terminals or neurosecretory cells for Ca²⁺-regulated exocytosis. Note that T254 is highly conserved among “presynaptic” syntaxin 1A, 2, and 3A molecules. We call all other syntaxin orthologs shown here “constitutive” syntaxins because they are used for constitutive secretion on the plasma membrane (PM) and intracellular compartments, such as the endosome and the lysosome, the <i>cis</i> and <i>trans</i> Golgi network (Golgi network), and endoplasmic reticulum (ER) [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0050072#pbio-0050072-b001" target="_blank">1</a>]. The yeast plasma membrane syntaxin orthologs SSO1 and SSO2, and syntaxins 4 and 131 from <i>Arabidopsis</i> are also shown here. (A more complete alignment can be see in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0050072#pbio-0050072-sg001" target="_blank">Figure S1</a>.) Unlike the synaptic syntaxins, syntaxin 4 and most syntaxin 11s have a valine (V) at the 254 equivalent position, syntaxins 6, 7, 12, 16, and 17 a leucine (L), and syntaxin 5 an isoleucine (I). The isoleucine found in the <i>syx^3–69</i> mutant resembles some of the wild-type syntaxin orthologs used for constitutive secretion. The core complex layers from 0 to +8 are identified at the bottom. The aa sequence was obtained from the NIH's National Center for Biotechnology Information (NCBI; <a href="http://www.ncbi.nlm.nih.gov" target="_blank">http://www.ncbi.nlm.nih.gov</a>) and aligned using the software DNAStar.</p></div